Near infrared imaging agent

ABSTRACT

This invention relates to an in-vivo diagnostic method based on near infrared radiation (NIR radiation) that uses water-soluble dyes and their biomolecule adducts, each having specific photophysical and pharmaco-chemical properties, as a contrast medium for fluorescence and transillumination diagnostics in the NIR range, to new dyes and pharmaceuticals containing such dyes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to an in-vivo diagnostic method based on nearinfrared radiation (NIR radiation) that uses water-soluble dyes andtheir biomolecule adducts, each having specific photophysical andpharmaco-chemical properties, as a contrast medium for fluorescence andtransillumination diagnostics in the NIR range, to new dyes andpharmaceuticals containing such dyes.

2. Description of Related Art

Diagnosability of diseases is very much dependent on obtaininginformation about the structures, as well as changes, of tissues of theprofound layers that are not primarily accessible. In addition topalpating, exposing or puncturing these tissues, such information can begained using sophisticated imaging methods such as x-raying, magneticresonance tomography, or ultrasonic diagnosis.

As biological tissue shows a relatively high permeability for long wavelight in the range of 650-1000 nm, a diagnostician can therefore use acompletely different method of tissue imaging. The fact that light inthe near infrared range can permeate through several centimeters oftissue is utilized in transillumination imaging. This technique as yetfacilitates diagnosis of inflammations of the paranasal and maxillarysinuses as well as the detection of accumulated fluids or blood insuperficial zones of tissue (Beuthan, J., Müller, G.;Infrarotdiaphanoskopie, Med. Tech. 1 (1992) 13-17).

Attempts at detecting breast tumours have been unsatisfactory so far(Navarro, G. A.; Profio, A. E.; Contrast in diaphanography of thebreast; Med. Phys. 150 (1988) 181-187; Aspegren, K.; Light ScanningVersus Mammography for the Detection of Breast Cancer in Screening andClinical Practice, Cancer 65 (1990) 1671-77) but there may be betterresults in the future due to most recent engineering progress(Klingenbeck, J.; Laser-Mammography with NIR-Light,Gynäkol.-Geburtsch.-Randsch. 33 Suppl.1 (1993) 299-300); Benaron, D. A.;Optical Imaging reborn with technical advances, Diagnostic Imaging(1994) 69-76).

In addition to detecting non-absorbed radiation, fluorescence radiationemitted after near infrared light treatment can provide tissue-specificinformation. This so-called autofluorescence is used to distinguishatherosclerotic and normal tissue (Henry, P. D. et al., Laser-InducedAutofluorescence of Human Arteries, Circ. Res. 63 (1988) 1053-59).

The main problem of applying near infrared radiation is theextraordinarily wide scattering of the light which permits only a ratherblurred image of a clearly contoured object despite differentphotophysical properties. The problem increases the greater the distancefrom the surface is and may be considered the major limiting factor ofboth transillumination and detection of fluorescence radiation.

Suitable fluorescent dyes that accumulate in diseased tissue (above all,in tumours) and that show a specific absorption and emission behaviour,may contribute towards enhancing the distinction of healthy fromdiseased tissue. The change caused by absorbing irradiated (scattered)light, or fluorescence induced by exciting radiation, is detected andprovides the actual tissue-specific information.

Examples of using dyes for in-vivo diagnostics in humans are photometricmethods of tracing in the blood to determine distribution areas, bloodflow, or metabolic and excretory functions, and to visualize transparentstructures of the eye (ophthalmology). Preferred dyes for suchapplications are indocyanine green and fluorescein (Googe, J. M. et al.,Intraoperative Fluorescein Angiography; Ophthalmology, 100 (1993),1167-70).

Indocyanine green (Cardiogreen) is used for measuring the liverfunction, cardiac output and stroke volume, as well as the blood flowthrough organs and peripheral blood flows (I. Med. 24 (1993) 10-27); inaddition, they are being tested as contrast media for tumour detection.Indocyanine green binds up to 100% to albumin and is mobilized in theliver. Fluorescent quantum efficiency is low in a hydrous environment.Its LD₅₀ (0.84 mmol/kg) is great enough; strong anaphylactic responsesmay occur. Indocyanine green is unstable when dissolved and cannot beapplied in saline media because precipitation will occur.

Photosensitizers designed for use in photodynamic therapy (PDT)(including haematoporphyrin derivatives, photophrin II, benzoporphyrins,tetraphenyl porphyrins, chlorines, phthalocyanines) were used up to nowfor localizing and visualizing tumours (Bonnet, R.; New photosensitizersfor the photodynamic therapy of tumours, SPIE Vol. 2078 (1994)). It is acommon disadvantage of the compounds listed that their absorption in thewavelength range of 650-1200 nm is only moderate. The phototoxicityrequired for PDT is disturbing for purely diagnostic purposes. Otherpatent specifications dealing with these topics are: U.S. Pat. No.4,945,239; WO 84/04665, WO 90/10219, DE 4136769, DE 2910760.

U.S. Pat. No. 4,945,239 describes a great number of equipmentarrangements for detecting breast cancer using transillumination andmentions the known fluorescein, fluorescamin, and riboflavin ascontrast-improving absorption dyes. These dyes share the disadvantagethat they absorb in the visible wavelength range of 400-600 nm in whichlight transmission capacity of tissue is very low.

DE 4136769 describes an apparatus for detecting fluorescence of tissueareas enriched with fluorescent substances. These substances arebacterial chlorophyll and its derivatives, and naphthalocyanines. Thesestructures show absorptions in the range of 700-800 nm at absorbencyindices of up to 70000 1 mol⁻¹ cm⁻¹. In addition to their fluorescentproperties, the compounds mentioned here are capable of generatingsinglet oxygen by radiation, thus having a cytotoxic effect(photosensitizers for photodynamic therapy). This photosensitizingactivity is highly undesirable for a pure, inactive diagnostic agent.

Furthermore, synthesis of bacterial chlorophyll compounds is expensiveand requires much effort as natural products have to be used as parentsubstances; the naphthalocyanines, however, frequently show a very lowphotostability. The known compounds of these classes are hardly solublein water, and synthesizing uniform hydrophilic derivatives is costly.

WO 84/04665 describes an in-vivo method for the fluorescence detectionof tumours using the following photosensitizers: haematoporphyrin andits derivative (Hp and HpD), uro- and copro- and protoporphyrin as wellas numerous mesosubstituted porphyrins, and dyes such as riboflavin,fluorescein, acridine orange, berberine sulfate and tetracyclines. Thephotophysical and pharmacochemical requirements mentioned above are notmet by said substances.

Folli et al., Cancer Research 54, 2643-2649 (1994), describe amonoclonal antibody connected with a cyanine dye that was used fordetecting a tumour implanted subcutaneously. Detection of profounderpathologic processes, however, requires much improved dyes. Higher dyedosages render the use of antibodies as carriers unsuitable in view ofthe side effects to be expected.

Cyanine dyes and polymethine dyes related to them are also used asphotographic layers. Such dyes need not have any luminescent properties.Cyanine dyes that have luminescent (fluorescent) properties have beensynthesized for use in fluorescent microscopy and flow cytometry andcoupled with biomolecules such as compounds containing iodine acetylgroups as specific labeling reagents for sulfhydryl groups of proteins(Waggoner, A. S. et al., Cyanine dye Labeling Reagents for SulfhydrylGroups, Cytometry, 10, (1989), 3-10). Proteins are labeled and isolatedin this way. More references: Cytometry 12 (1990) 723-30; Anal. Lett. 25(1992) 415-28; Bioconjugate Chem. 4 (1993) 105-11.

DE 39 12 046 by Waggoner, A. S. describes a method for labelingbiomolecules using cyanine and related dyes such as merocyanine andstyryls that contain at least one sulfonate or sulfonic acid grouping.This specification relates to a single and two-step labeling method in ahydrous environment, with a covalent reaction taking place between thedye and the amine, hydroxyl, aldehyde or sulfhydryl group on proteins orother biomolecules.

DE 3828360 relates to a method for labeling antitumour antibodies, inparticular, antibodies specific to melanoma and colonic cancer, usingfluorescein and indocyanine green for ophthalmologic purposes. Bondingof indocyanine green to biomolecules is not covalent (dye-antibodycombination, mixture).

The known, state-of-the-art methods of in-vivo diagnosis using NIRradiation thus show a number of disadvantages that prevented their wideapplication in medical diagnostics.

Direct use of visible light or NIR radiation is restricted tosuperficial body zones, which is due to the widely scattered incidentlight.

Adding dyes to improve contrast and resolution, however, gives rise to anumber of other problems. The dyes should meet the requirements thatgenerally apply to diagnostic pharmaceuticals. As these substances aremostly applied at higher doses and for a longer diagnostic period, theyshould be low-toxic. In addition, dyes suitable for diagnostic purposesshould be well soluble in water and sufficiently stable in chemical andphotophysical respect, at least for as long as the diagnostic periodlasts. Stability as regards metabolization in the system is alsodesirable.

So far, neither dyes nor a suitable method for in-vivo diagnosis usingNIR radiation has been available.

It is therefore an object of this invention to provide a method ofin-vivo diagnosis that overcomes the disadvantages of prior art.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows fluorescent light images (black-and-white) of a mouse(Swiss Nude) at various points in time after i.v. application of 3.8mmol/kg body weight of2-[7-[1,3-dihydro-3,3-dimethyl-5-(methoxycarbonyl)-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-(methoxycarbonyl)-1-(4-sulfobutyl)-3H-indolium,sodium salt. Photographs A-E are right lateral images, and photograph Fis a posterior image, taken at the following times:

-   -   A: prior to application,    -   B: 30 seconds after application,    -   C: 1 minute after application,    -   D: 10 minutes after application,    -   E: 1 hour after application, and    -   F: 18 hours after application.

SUMMARY OF THE INVENTION

This problem is solved according to the invention by providing a methodof in-vivo diagnosis using NIR radiation in which compounds of thegeneral formula IB _(l)−(F−W _(n))_(m)   (I)are used,

wherein 1 represents a number from 0 to 6, n a number from 0 to 10, andm a number from 1 to 100,

B is a biological detecting unit having a molecular weight of up to30000 that bonds to specific cell populations or selectively toreceptors, or accumulates in tissues or tumours, or generally stays inthe blood, or is a macromolecule that bonds non-selectively;

F represents a dye showing maximum absorption in the range of 650 to1200 nm;

W represents a hydrophilic group that improves water-solubility, withthe n-octanol-water distribution coefficient of the compound accordingto formula I being less than or equal to 2.0 for 1=0;

as well as their physiologically tolerable salts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes compounds of the general formula IB _(l)−(F−W _(n))_(m)   (I)are used,

wherein

the letter l represents a number from 0 to 6, n represents a number from0 to 10, and m represents a number from 1 to 100,

B is a biological detecting unit having a molecular weight of up to30,000 that bonds to specific cell populations or selectively toreceptors, or accumulates in tissues or tumours, or generally stays inthe blood, or is a macromolecule that bonds non-selectively;

F represents a dye showing maximum absorption in the range of 650 to1200 nm; and

W represents a hydrophilic group that improves water-solubility, withthe n-octanol-water distribution coefficient of the compound accordingto formula I being less than or equal to 2.0 for L=0;

as well as their physiologically tolerable salts.

Compounds of the general formula I that are particularly well-suited forthe method according to the invention are those in which, for example, Bis an amino acid, a peptide, CDR (complementarity determining region),an antigen, a hapten, an enzyme substrate, an enzyme cofactor, biotin, acarotinoid, a hormone, a neurohormone, a neurotransmitter, a growthfactor, a lymphokin, a lectin, a toxin, a carbohydrate, anoligosaccharide, a polysaccharide, a dextrane, an oligonucleotide or areceptor-bonding pharmaceutical.

Furthermore, such compounds of the general formula I are particularlyappropriate for the method according to the invention in which, forexample, F represents a cyanine dye of the general formula IIa

wherein

r represents the numbers 0, 1 or 2, on condition that, for r=2, therespective fragments L⁶ and L⁷ that occur in duplicate may be the sameor different,

L¹ to L⁷ are same or different, each independently representing afragment CH or CR, where

-   -   R is a halogen atom, a hydroxy, carboxy acetoxy, amino, nitro,        cyano or sulfonic acid group or an alkyl, alkenyl, hydroxyalkyl,        carboxyalkyl, alkoxy, alkoxycarbonyl, sulfoalkyl, alkylamino,        dialkylamino or halogenalkyl residue containing up to 6 carbon        atoms, an aryl, alkylaryl, hydroxyaryl, carboxyaryl, sulfoaryl,        arylamino, diarylamino, nitroaryl or halogenaryl residue        containing up to 9 carbon atoms, or where R represents a bond        that bonds to another residue R and forms a 4- to 6-member ring        together with the interspersed residues L¹ to L⁷, or where R        represents one bond, respectively, at two different positions        that are linked via a —CO— fragment,

R3 to R¹² are same or different, each independently representing ahydrogen atom, a residue B or W as defined above, or an alkyl or alkenylresidue containing up to 6 carbon atoms or an aryl or aralkyl residuecontaining up to 9 carbon atoms, said alkyl, alkenyl, or aryl or aralkylresidue optionally carrying an additional residue W as defined above, orto each pair of adjacent residues R³ to R¹⁰ are annealed 5- to 6-memberrings that may be saturated, unsaturated or aromatic, and that mayoptionally carry an additional residue R as defined above, with dueregard for the interspersed C atoms,

X and Y are same or different, each independently representing an O, S,Se or Te or a —C(CH₃)₂—, —CH=CH— or —CR¹³R¹⁴— fragment,

where R¹³ and R¹⁴ independently represent a hydrogen atom, a residue Bor W as defined above, or an alkyl or alkenyl residue containing up to 6carbon atoms or an aryl or aralkyl residue containing up to 9 carbonatoms, the alkyl, alkenyl, aryl or aralkyl residue optionally carryingan additional residue W as defined above, represents a squarain dye ofthe general formula II b

wherein

s and t independently represent the numbers 0 or 1 on the condition thats and t do not represent the number 1 at the same time,

and R³ to R ¹², x and y are as defined above,

represents a styryl dye of the general formula II c

wherein

r, L¹ to L⁶, R³ to R¹¹ and X are as defined above,

or represents a merocyanine dye of the general formula II d

wherein

r, L¹ to L⁶, R³ to R⁸, R¹¹ and X are as defined above and G representsan oxygen or sulfur atom.

Such compounds of the general formula I are particularly appropriate forthe method according to the invention in which, for example, W is acarboxy or sulfonic acid group or a carboxyalkyl group or analkoxycarbonyl group or an alkoxyoxoalkyl group containing up to 12carbon atoms,

represents a residue of the general formulas III—(CH₂)_(a)—O—Z or (—CH₂—CH₂—O)_(a)—Z   (III)

wherein

a represents the numbers 0 to 6

Z comprises a hydrogen atom or an alkyl residue containing 3 to 6 Catoms that includes 2 to n-1 hydroxy groups, with n being the number ofC atoms, or an aryl or aralkyl residue containing 6 to 10 C atoms andcarrying 2 to 4 additional hydroxy groups, or an alkyl residuecontaining 1 to 6 C atoms and carrying 1 to 3 additional carboxy groups,or an aryl residue containing 6 to 9 C atoms and carrying 1 to 3additional carboxyl groups, or an aralkyl residue or a nitroaryl or anitroaralkyl residue containing 6 to 15 C atoms, or a sulfoalkyl residuecontaining 2 to 4 C atoms carrying 1 to 3 additional carboxy groups,

or represents a residue of the general formulas III a or III b

or a residue of the general formula IIIc

-   -   —(CH₂)_(o)—(CO)_(p)—NR¹—(CH₂)—(NH—CO)_(q)—R²   (IIIc)    -   wherein    -   o and s independently represent the numbers 0, 1, 2, 3, 4, 5 or        6,    -   p and q independently represent 0 or 1,    -   R¹ and R² independently represent a residue Z as defined above        except the substituents of the general formulas IIIa and IIIb,        or independently represent a residue of the general formulas        IIId or IIIe    -   on the condition that p and q=1,

or represents a residue of the general formula IIIc as defined above.

The compounds used for the method according to the invention arecharacterized in that they absorb and fluoresce in the wavelength rangeof 650 to 1200 nm, have absorption coefficients of approx. 100 000100,000 1 mol⁻¹ cm⁻¹ and more and, where fluorescence is desirable, havea fluorescence quantum efficiency greater than 5%, are sufficientlywater-soluble, tolerable and stable in vitro and in vivo as well asphotostable. They are discharged as completely as possible in as short atime as possible. The compounds used according to the invention aresynthesized easily and at a favorable price in only a few reaction stepsfrom parent materials that are available on the market.

When applying the method according to the invention in in-vivodiagnosis, one or several substances of the general formula I is/areadministered to the tissues, for example, by intravenous injection, thenthey are irradiated with light from the visible to the near infraredrange of 650 to 1200 nm. Radiation that is not absorbed and fluorescenceradiation are recorded separately or simultaneously, or against eachother with a delay. A synthetic image is generated from the dataobtained.

Fluorescent images can be recorded using various methods. Preferred arethose methods where the tissue is irradiated extensively, fluorescenceinformation is visualized in local resolution by a CCD camera, or wherethe tissue sectors to be imaged are scanned by a light ray concentratedin a fibre optical waveguide and signals obtained are converted into animage by computing. The light is beamed in in the narrow-band range atwavelengths close to the maximum absorption or at fluorescence-excitingwavelengths of the compounds of the invention. Radiation that was notabsorbed can be recorded as described, and signals obtained beprocessed.

The irradiation angle and the angle of observation can be selected fromcase to case to meet anatomic and optimum contrast requirements. Thesensitivity of the method may be improved by subtracting the imagesprior to and after administering the dye. Evaluating the time curve ofdye-related changes may reveal useful additional information for thediagnosis.

The measurement methods used are known to a person skilled in the art.The expert will also know what equipment parameters should be set toobtain optimum recording and evaluation conditions at given absorptionor fluorescence wavelengths of the dyes of the general formula I usedaccording to the invention.

The compounds of the general formula I used for the method of theinvention cover a wide range of exciting and emission wavelengths due tothe variable structure of the dye system F. It is possible to gainproducts with exciting wavelengths that correspond to a specific sourceof excitation, e.g. at the diode laser unit, and are therefore adaptedto a given measuring system or equipment component.

The techniques described even permit localization of small objectshaving a volume of only a few mm³ at the profounder layers of tissue orin non-transparent body fluids. Due to light scattering and the limitedresolution it entails it is still difficult to determine the exact shapeand size of such objects but this is not required to solve someimportant diagnostic questions.

Surprisingly, a fluoroscopic image of a mouse (Swiss Nude) taken afterapplying a cyanine dye using a CCD camera showed a 1000 times greaterfluorescent intensity as compared to a similarly dosed porphyrin.

The method described that uses the compounds of the invention isparticularly suitable for the visualization of tissue withoutpathological alterations, systemic diseases, tumours, blood vessels,atherosclerotic plaques, perfusion and diffusion.

The compounds used according to the invention are applied to the tissuein different ways. Intravenous administration of the dyes isparticularly preferred.

Dosage may be quite different depending on the purpose of application.The goal to be achieved is a detectable concentration of dye in thetissue zone to be diagnosed, for which a concentration of 1-100 μg/ml inthe tissue or in body fluids will mostly be sufficient. Thisconcentration is reached by direct injection into small body cavities orsmall blood or lymph vessels, normally by applying 0.1-100 mg of therespective dye contained in 0.1 to 10 ml of vehicle liquid. In thiscase, 1 to 10 mg of dye are preferred. Higher doses are mostly requiredto stain blood vessels or to detect specific tissues or structures afterintravenous injection (greater than or equal to 100 mg). The upper limitof dosage is only set by the tolerability of the respective substancesand preparations.

Thus, the invention relates to the use of compounds of the typeB_(l)−(F−W_(n))_(m), in which F represents a dye from the class ofpolymethine dyes, in particular, cyanine dyes. Merocyanine, styryl,oxonol and squarilium dyes may also be used. W is a structural elementthat contributes essentially to the hydrophilia of the whole molecule.Particularly preferred are compounds in which l represents the number 0,with their n-octanol/water distribution coefficient being smaller than 2(n-octanol/0.01 M TRIS buffer containing 0.9% of sodium chloride, set topH 7.4, both phases saturated against each other).

A biological detecting unit B may, for example, be an amino acid, apeptide, a CDR (complementarity determining regions), an antigen, ahapten, an enzyme substrate, an enzyme cofactor, biotin, a carbotinoid,a hormone, neurohormone, neurotransmitter, a growth factor, a lymphokin,a lectin, a toxin, a carbohydrate, an oligosaccharide, a polysaccharide,a dextran, an oligonucleotide made resistant to nucleases or areceptor-bonding pharmaceutical.

Compounds from the above-mentioned groups include, for example,oxytocins, vasopressins, angiotensins, melanocyte-stimulating hormones,somatostatins, tyrotropin-releasing hormones, gonadotropin-releasinghormones, testosterones, estradiols, progesterones, cortisols,aldosterones, vitamin D, gastrins, secretins, somatropins, insulins,glucagons, calcitonin, STH-releasing hormones, prolactins, encephalins,dopamines, noradrenalines, serotonins, epinephrines, interleukines,angiogenins, thymopoietins, erythropoietins, fibrinogens,angiotensinogens, mecamylamines, ranitidine, cimetidine, lovastatines,isoproterenol derivatives or transferrin.

These substances facilitate accumulation in specific parts of the bodyby targeting the biological detecting unit through certain mechanisms.These mechanisms include bonding to extracellular structures,accumulation through various biological transport systems, recognitionof cell surfaces or recognition of intracellular components.

Other compounds can be used according to the invention in which B is anon-selectively bonding macromolecule, such as polylysine, polyethyleneglycol, methoxypolyethylene glycol, polyvinyl alcohol, dextran,carboxydextran or a cascade polymer-like structure that is covalentlybonded to F.

The alkyl-, aryl- or aralkyl residue with hydroxy groups contained inthe compounds of the general formula I used according to the invention,are for example, 2-hydroxyethyl-, 2-hydroxypropyl-, 3-hydroxypropyl-,4-hydroxybutyl-, 2,3-dihydroxypropyl-, 1,3-dihydroxyprop-2-yl-,tris-(hydroxymethyl)-methyl-, 1,3,4-trihydroxybut-2-yl-glucosyl-,4-(1,2-dihydroxyethyl)phenyl- or 2,4-, 2,5-, 3,5- or 3,4-dihydroxyphenylresidues.

An alkyl-, aryl- or aralkyl residue containing 1 to 3 carboxy groups maybe, for example, a carboxymethyl-, carboxyethyl-, carboxypropyl-,carboxybutyl-, 1,2-dicarboxyethyl-, 1,3-dicarboxypropyl-,3,5-dicarboxyphenyl-, 3,4-dicarboxyphenyl-, 2,4-dicarboxyphenyl or4-(1,2-dicarboxyethyl)-phenyl residue.

A sulfoalkyl residue preferably is a 2-sulfoethyl-, 3-sulfopropyl- and4-sulfobutyl residue.

Compounds in which W takes the position of R⁴ or R⁸, R⁶ or R¹⁰ and R¹¹or R¹², and is also present in duplicate at positions R³/R⁵ or R⁷/R⁹ areparticularly preferred.

The dyes used according to the present invention absorb in the spectralrange from 650 nm to 1200 nm. The absorption coefficients of thecompounds are ca. 100,000 1 mol⁻¹ cm⁻¹ and more for one dye molecule.Fluorescent quantum efficiencies are greater than 5% for all dyes usedfor fluorescent imaging.

Another object of this invention are cyanine dyes of the general formulaV.

where

Q represents a fragment

-   -   where R³⁰ represents a hydrogen atom, a hydroxy group, a carboxy        group, an alkoxy residue containing 1 to 4 carbon atoms or a        chlorine atom, b is an integer (2 or 3), R³¹ represents a        hydrogen atom or an alkyl residue containing 1 to 4 carbon        atoms,

X and Y independently represent an —O—, —S—, —CH=CH— or—C(CH₂R³²)(CH₂R³³)— fragment each,

R20 to R²⁹, R³² and R³³ independently represent a hydrogen atom, ahydroxy group, a carboxy-, a sulfonic acid residue or a carboxyalkyl-,alkoxycarbonyl or alkoxyoxoalkyl residue containing up to 10 C atoms ora sulfoalkyl residue containing up to 4 C atoms,

or a non-selectively bonding macromolecule or a residue of the generalformula VI—(O)_(v)—(CH₂)_(o)—CO—NR³⁴—(CH₂)_(s)—(NH—CO)_(q)—R³⁵   (VI)

on the condition that, where X and Y are O, S, —CH=CH— or —C(CH₃)₂—, atleast one of the residues R20 to R29 corresponds to a non-selectivelybonding macromolecule or a compound of the general formula VI,

-   -   where    -   o and s equal 0 or independently represent an integer between 1        and 6,    -   q and v independently represent 0 or 1,    -   R³⁴ represents a hydrogen atom or a methyl residue,    -   R³⁵ represents an alkyl residue containing 3 to 6 C atoms and        comprising 2 to n-1 hydroxy groups, with n being the number of C        atoms, or an alkyl residue containing 1 to 6 C atoms that        carries 1 to 3 additional carboxy groups, an aryl residue        containing 6 to 9 C atoms or arylalkyl residue containing 7 to        15 C atoms, or a residue of the general formula IIId or IIIe    -   on the condition that q is 1,    -   or a non-selectively bonding macromolecule,

R²⁰ and R²¹, R²¹ and R²², R²² and R²³, R²⁴ and R²⁵, R²⁵ and R²⁶, R²⁶ andR²⁷, together with the interspersed carbon atoms, form a 5- or 6-memberaromatic or saturated annelled ring,

as well as their physiologically tolerable salts.

In the compounds according to the invention of the general formula V,the alkyl-, aryl- or aralkyl residues containing hydroxy- or carboxygroups have the preferred composition as defined above.

The following are particularly preferred cyanine dyes:

-   -   5-[2-[(1,2-dicarboxyethyl)amino]-2-oxoethyl]-2-[7-[5-[2-[(1,2-dicarboxyethyl)amino]-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium,        inner salt, potassium hydrogen salt;    -   2-[7-[5-[2-[(11-carboxy-2-oxo-1,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-1-undecyl)amino]-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-ethyl-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium,        inner salt;    -   2-[7-[1,3-dihydro-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)-amino]-2-oxoethyl]-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)amino]-2-oxoethyl]-1-(4-sulfobutyl)-3H-indolium,        sodium salt;    -   2-[7-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-(methoxypolyoxyethylene)aminocarbonyl-1-(4-sulfobutyl)-3H-indolium,        sodium salt;    -   3-(3-carboxypropyl)-2-[7-[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3-methyl-1-(4-sulfobutyl)-3H-indolium,        sodium salt;    -   2-[[3-[[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulfobutyl)2H-indol-2-yliden]methyl]-2-hydroxy-4-oxo-2-cyclobuten-1-yliden]methyl]-1,1-dimethyl-3-ethyl-1H-benz(e)indolium,        inner salt;    -   2-[7-[1,3-dihydro-5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium,        sodium salt.

Another important characteristic of the compounds of the invention andthe compounds used according to the invention is their hydrophiliamarked by an n-octanol/water distribution coefficient smaller than 2.0(distribution coefficient n-octanol/0.01 M TRIS buffer containing 0.9%sodium chloride, set to pH 7.4, both phases saturated against eachother). The compounds do not have any distinct photosensitizing orphototoxic properties that would be undesirable in a diagnostic reagent.They are tolerated well, and discharged.

The hydrophilic behaviour of the compounds of the invention make themdiffer from dyes that have been proposed for use in in-vivo diagnostics.Especially with cyanine dyes, fluorescent quantum efficiency values, dueto aggregation, drop dramatically in a hydrous environment, and arecomparable to values measured in non-polar solvents; increasedsolubility in water and the space requirements of the hydrophilic groupssuppress the formation of aggregates and micelles.

A group of preferred compounds shows little protein affinity; itspharmacokinetic behaviour is similar to that of insulin or saccharose,for example.

Surprisingly, these compounds showed diagnostically sufficientaccumulation in specific structures of the system, e.g. in tumours,despite their simple molecular structure. When the dye has spreadequally throughout the organism, its elimination, compared to thesurrounding tissue, is delayed in tumour zones.

Tolerance of the substances is very good. Substances having LD₅₀ valuesgreater than 0.5 mmol/kg body weight referred to a single dye molecule,are particularly preferred.

The compounds of the invention and the compounds used according to theinvention are characterized by great in-vitro and in-vivo stability, aswell as photostability. When the aqueous solution is allowed to stand ina daylit room, 98% of each of the compounds that are particularlypreferred show no changes after 2 days, 70% show no changes after 12days.

The photophysical and pharmacokinetic properties described of thecompounds of the invention and the compounds used according to theinvention, also differ from those of the only cyanine dye approved forapplication in humans: indocyanine green (Cardiogreen).

Another object of the present invention are compounds of the generalformula I in which the l-values of B are greater than or equal to 1,preferably 1 or 2.

Cyanine dyes can be synthesized that have a great extinction coefficientwhen absorbing light at wavelengths from 650 to 120 nm, and thatfluoresce with great efficiency. Cyanine dyes of the invention andcyanine dyes used according to the invention are mainly synthesizedaccording to methods known from the literature, for example F. M. Hamerin The Cyanine Dyes and Related Compounds, John Wiley and Sons, NewYork, 1964; Cytometry 10 (1989) 3-10; 11 (1990) 418-430; 12 (1990)723-730; Bioconjugate Chem. 4 (1993) 105-11, Anal. Biochem. 217 (1994)197-204; Tetrahedron 45 (1989) 4845-66, European Patent No. EP 0 591 820A1.

The dye-biomolecule adducts of the general formula I used according tothe invention are prepared by reacting a known compound F—W_(n) preparedaccording to the methods mentioned above with a biological detectingunit B.

The compound F—W_(n) should therefore contain at least one, preferablyexactly one, grouping that can react covalently to an amine, hydroxy,aldehyde or sulfhydryl group on the biological detecting units. Suchgroupings are known from the literature and described in some detail,for example, in DE 39 12 046.

Particularly preferred are isothiocyanate, isocyanate, andhydroxysuccinimide ester or hydroxysulfosuccinimide ester groupings thatare reactive to amino functions and form a thiourea, urea and amidebridge, as well as halogenacetyl and succinimide groupings that arereactive to sulfhydryl groups and form a thioester bridge.

Furthermore, carboxy groups with alcoholic functions may form esterlinkages or ether structures using appropriate activating reagents (e.g.DCC), and aldehyde functions combined with hydrazines may result inimine structures.

The reactive groupings mentioned are added to the dyes of the inventionor dyes used according to the invention of the general formula I ortheir synthetic predecessors, or existing function groups are convertedinto the reactive groupings. The reactive groupings may be directlybonded to the dye system via so-called linker structures (e.g. alkylchains, aralkyl structures).

The F—W_(n) compounds are preferably reacted with the biologicaldetecting units B in DMF or a hydrous environment or DMF/water mixturesat pH values between 7.4 and 10. The molar proportion of dye andbiomolecule (charging ratio) is determined using absorptionspectrometry. Components that are not bound are separated bychromatography or filtering.

Macromolecules that have the appropriate function groups may be coupledto the dyes in a similar way.

The substances may have quite different properties. Their molecularweight may be from a few hundreds to more than 100,000. The substancescan be neutral or electrically charged. Salts of acid dyes andphysiologically acceptable bases, such as sodium, methyl glutamine,lysine, or salts containing lithium, calcium, magnesium, gadolinium inthe form of cations.

The dye-biomolecule adducts thus gained excellently meet the abovephotophysical, toxicological, chemical and economic requirements.

Another object of the present invention is the use of cyanine dyes ofthe general formula V for in-vivo diagnosis using NIR radiation, inanalogy to the use of compounds of the general formula I.

Yet another object of the present invention are diagnostic reagents thatcontain compounds of the general formulas V or I.

These reagents are produced according to methods known to personsskilled in the art, optionally by adding common adjuvants, diluents andthe like. This includes physiologically tolerable electrolytes, buffers,detergents and substances for adjusting osmolarity and for improvingstability and solubility such as cyclodextrine. Sterility of thepreparations during their production and, in particular, before theirapplication is to be ensured by taking the steps common inpharmaceutics.

The invention will now be explained by the following examples.

EXAMPLES Example 1

Preparation of5-[2-[[(1,2-dicarboxyethyl)amino]-2-oxoethyl]-2-[7-[5-[2-(1,2-dicarboxyethyl)amino]-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium,inner salt, potassium hydrogen salt.

Di-N-hydroxysuccinimide ester is prepared from5-carboxymethyl-2-[7-[5-carboxymethyl-1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium,inner salt, potassium hydrogen salt according to known methods(Cytometry 11 (1990) 418-430).

0.16 g (1.22 mmol) of aspartic acid in 1 ml of DMF are added to asolution of 0.5 g (0.51 mmol) of the disuccinimidyl ester in 5 ml DMF.The reaction mixture is stirred at room temperature for 48 h. Theproduct is precipitated by adding ether, purified on RP-18 (LiChroprep,15-25 μ, H₂O:MeOH 99:1 to 1:1) and lyophilized. 0.27 g (51%) of productare gained after drying for 24 hours at 50° C./0.01 mbar.

Analysis: Calc.: C 54.43 H 5.54 N 5.40 O 24.68 S 6.18 K 3.77 Det.: C54.04 H 5.81 N 5.22 S 6.13 K 3.85

Example 2

Preparation of2-[7-[5-[2-[(11-carboxy-2-oxo-1,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-1-andecyl)amino]-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-ethyl-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium,inner salt.

43 mg (0.65 mmol) of 85% hydrazine hydrate in 1 ml of methanol areslowly added by dropping to a solution of 0.5 g (0.73 mmol)2-[7-[5-(carboxymethyl)-i,3-dihydro-3,3-dimethyl-1-ethyl-2H-indol-2-yliden]-1,3,5-hepta-trienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium-N-succinimidylester, inner salt in 5 ml of methanol (Cytometry 11 (1990) 418-430) at−10° C. and stirred for 2 hours at this temperature. The reactionmixture is evaporated under a vacuum to approx. 3 ml mixed with 1 ml ofisopropanol and kept overnight at −20° C. The crystals that precipitateare sucked off and dried using the oil pump. The yield is 0.27 g (61%)of tricarbocyanine carbonic acid hydrazide.

0.27 g (0.45 mmol) of the hydrazide is added under stirring to asolution of 0.21 g (0.51 mmol) of diethylene triamine pentaaceticmonoethyl ester monoanhydride in 20 ml DMF and 0.2 ml triethylamine. Themixture is kept agitated at room temperature for 48 hours. The solventis evaporated at 0.2 mbar after filtering, the residue is mixed up withCH₂Cl₂, filtered off, and dried under high vacuum. The product gained isstirred up in 5 ml of 3 M aqueous NaOH at room temperature for 4 hours.Then, a pH value of 2.0 is set using semiconcentrated HCl. 1 ml ofisopropanol is added. After allowing the mixture to stand at 4° C. for18 hours, crystals that have precipitated are sucked off and dried underhigh vacuum at 60° C. for 24 hours.

Yield: 0.23 g (52%) of granulate that glimmers dark red.

Analysis: Calc.: C 59.32 H 6.60 N 9.88 O 20.96 S 3.23 Det.: C 54.15 H6.70 N 9.50 S 3.19

Example 3

Preparation of2-[7-[1,3-dihydro-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)amino]-2-oxoethyl]-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)amino-2-oxoethyl]-1-(4-sulfobutyl)-3H-indolium,sodium salt.

A solution of 0.08 mmol of the N,N-disuccinimidyl ester from Example 1in 1 ml of DMF is added to a solution of 800 mg ofmethoxypolyoxyethylene amine (ca. 0.16 mmol; average molar weight ca.5000) in 10 ml of CH₂Cl₂ and kept agitated at room temperature for 24hours. The solid product that precipitates after adding ether isfiltered off and purified by chromatography (Sephadex G5O medium, H₂O aseluent), yield approx. 58% of green-blue powder after lyophilization anddrying above P₂O₅.

Average molar weight calc.: 10771, det.: 10820.

Example 4

Preparation of2-[7-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-(methoxypolyoxyethylene)aminocarbonyl-1-(4-sulfobutyl)-3H-indolium,sodium salt.

0.41 g (0.5 mmol) of2-[7-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-hepatrienyl]-3,3-dimethyl-5-carboxy-1-(4-sulfobutyl)-3H-indolium-N-succinimidylester, sodium salt are stirred in an argon atmosphere together with 2.3g of methoxy polyoxyethylene amine (0.46 mmol; average molar weight:5000) in 70 ml CH₂Cl₂ at room temperature for 18 hours. The solvent isreduced by half under vacuum and the product is isolated as described inExample 3. The yield is 2.1 g of product in the form of a green bluepowder.

Average molar weight calc.: 5701, det.: 5795.

Example 5

Preparation of3-(3-carboxypropyl)-2-[7-[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3-methyl-1-(4-sulfobutyl)-3H-indolium,sodium salt.

6.5 g (50 mmol) of phenylhydrazine hydrochloride and 8.7 g (55 mmol) of5-methyl-6-oxoheptanoic acid are stirred in 50 ml of concentrated aceticacid at room temperature for 1 hour, and at 120° C. for 5 hours. Afterreducing by evaporation, the residue is mixed up with 20 ml of water,and the crystals that have precipitated are filtered off and dried usingthe oil pump.

This yields 9.6 g (83%) of brownish crystals that are suspended in 60 mlof dichlorobenzene and, after adding 11.6 g (85 mmol) of 1,4-butanesultone, heated for 8 hours to 150° C. After the mixture has cooled downto room temperature, 200 ml of acetone are added, and the precipitate isfiltered off. It is suspended in ether, filtered off again after 18hours of stirring, and dried using the oil pump. The yield is 10.7 g(70%) of 3-(3-carboxypropyl)-2,3-dimethyl-1-(4-sulfobutyl)-3H-indoleninwhich is purified by chromatography (RP-18, LiChroprep, 15-25 μ,MeOH:H₂O as eluent).

The indotricarbocyanine dye is prepared by heating 5.0 g (13.6 mmol) ofindolenin and 1.9 g (6.8 mmol) of glutaconaldehyde dianilhydrochloridein 100 ml of acetic anhydride for 30 minutes to 120° C., while adding 25ml of conc. acetic acid and 2.3 g (27.6 mmol) of anhydrous sodiumacetate. 500 ml of ether are added to the precipitate gained which ispurified by chromatography (in portions of 1.0 g, RP-18, LiChroprep,15-25 μ, MeOH:H₂O as eluent) and finally lyophilized. The yield is 2.5 g(45%) of the final product.

Analysis: Calc.: C 60.13 H 6.28 N 3.42 O 19.54 S 7.83 Na 2.81 Det.: C59.90 H 6.34 N 3.39 S 7.72 Na 2.78

Example 6

Preparation of2-[[3-[[3-(carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulfobutyl)2H-indol-2-yliden]methyl]2-hydroxy-4-oxo-2-cyclobuten-1-yliden]methyl]-1,1-dimethyl-3-ethyl-1H-benz(e)indolium,inner salt.

3.65 g (10.0 mmol) of 3-ethyl-1,1,2-trimethyl-lH-benz(e)indoliumiodideare added to a solution of 1.36 g (8.0 mmol) squaric diethyl ester and1.6 ml triethylamine in 12 ml of ethanol that is heated up to 70° C.After 10 minutes of stirring at 80° C., the mixture is cooled down toOC. The precipitated, red-coloured crystals are filtered off, washedwith ether, and dried under vacuum. Purification by chromatography onsilica gel (CH₂Cl₂:AcOH 9:1 to 7:3) yields 1.33 g (46%) of2-ethoxy-1-[(3-ethyl-1,1-dimethyl-1H-benz(e)indol-2-yliden)-methyl]-cyclobuten-3,4-dion.

This substance is suspended in 15 ml of boiling ethanol and mixed understirring with 0.5 ml of 40% NaOH. The solution gained is stirred for 5minutes at 80° C. and mixed with 5 ml of 2N HCl after cooling down toroom temperature. The1-[(3-ethyl-1,1-dimethyl-1H-benz(e)indol-2-yliden)-methyl]-2-hydroxycyclobutene-3,4-dion(1.30 g) that precipitates after evaporating is filtered, dried and usedfor the next step of synthesis without being purified.

The squarain dye is prepared by reacting 1.30 g (3.9 mmol) of thesquaric acid derivative gained with 1.43 g (3.9 mmol)of3-(3-carboxypropyl)-2,3-dimethyl-1-(4-sulfobutyl)-3H-indolenin. Thecomponents obtained are heated for 18 hours in 80 ml of toluene and 80ml 1-butanol at the water separator and then freed from solvents undervacuum. The residue is mixed with ether, and the crystals that haveformed are filtered off after 16 hours of stirring at room temperature,and purified by chromatography (RP-18, LiChroprep, 15-25 μ, MeOH:H₂O aseluent), yield: 0.95 g (36%).

Analysis: Calc.: C 68.60 H 6.20 N 4.10 O 16.40 S 4.70 Det.: C 68.25 H6.35 N 4.04 S 4.59

Example 7

Preparation of2-[7-[1,3-dihydro-5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium,sodium salt.

2.0 g (6.4 mmol) of 2,3,3-trimethyl-3H-indol-5-yl acetic succinimidylester in 50 ml CH₂CI₂ are mixed with 0.84 g (6.4 mmol) of4-aminomethyl-2,2-dimethyl-1,3-dioxolane. After 5 hours of stirring atroom temperature, the mixture is poured on 100 ml of water and extractedwith CH₂CL₂; the organic phases are evaporated. After chromatographicpurification (silica gel CH₂Cl₂:MeOH 98:2) 1.86 g (88%) of the amide aregained which are stirred for 12 hours in 20 ml of dichlorobenzene and1.36 g (10.0 mmol) of 1,4-butane sultone at room temperature, and at100° C. The granulate that is formed after stirring the mixture up with50 ml of acetone is filtered off and purified by chromatography (RP-18,LiChroprep, 15-25 μ, MeOH:H₂O as eluent). The yield is 0.85 g (28%referred to the parent compound) of5-[2-[(2,2-dimethyl-1,3-dioxa-4-cyclopentyl)methyl]amino-2-oxoethyl]-2,3,3-trimethyl-1-(4-sulfobutyl)-3H-indolenin.

The reaction to produce the dye is similar to Example 4. The substanceis heated for 10 minutes to 120° C. The crude product is stirred at roomtemperature in 5 ml MeOH by adding 100 mg toluene-p-sulfonic acid for 16hours; insoluble parts are separated. The filtrate is then kept at −20°C. after adding 3 ml of isopropanol. The powder that precipitates ispurified by chromatography (RP-18, LiChroprep, 15-25 μ, MeOH:H ₂O aseluent), lyophilized and dried for 24 hours at 50° C./0.01 mbar. Yield:0.32 g (37%).

Analysis: Calc.: C 56.70 H 6.45 N 5.88 O 20.14 S 6.73 K 4.10 Det.: C56.39 H 6.88 N 5.67 S 6.58 K 3.93

Example 8

2-[7-[1,3-dihydro-3,3-dimethyl-5-(methoxycarbonyl)-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3 -dimethyl-5-(methoxycarbonyl)-1-(4-sulfobutyl)-3H-indolium, sodiumsalt at a dose of 3.8 μmol/kg body weight was applied intravenously toan anaesthetized, tumour-bearing mouse (Swiss Nude; tumour LS 174 T atthe right hindleg).

The laser-induced fluorescent images were taken prior to, and at variouspoints in time after, applying the substance with a fluorescence imager(at Physikalisch-Technische Bundesanstalt, Berlin Charlottenburg).Radiation was excited using monochromatic laser light at 740 nm bydecoupling the radiation via a fibre optical waveguide system. Excitingradiation below 740 nm was removed using a cutoff filter. Thelaser-induced fluorescence light above 740 nm was recorded using a CCDcamera (Charge Coupled Device), and data was stored in the form ofblack-and-white images.

The sequence of shots shown in FIG. 1 clearly shows a general increaseof fluorescent intensity after the substance was applied (A, B). Auniform distribution of intensity can be observed after 30 seconds, withvalues being increased in the hepatic and pulmonary regions and in thetumour (B). With more time elapsing (up to 1 h) (C, D, E), the substancespreads more and more throughout the animal. After 18 hours, a clearlyincreased fluorescent intensity can be observed in the tumour (righthindleg) as compared with the rest of the body.

FIG. 1 shows fluorescent light images (black-and-white) of a mouse(Swiss Nude) at various points in time after i.v. application of 3.8mmol/kg body weight of2-[7-[1,3-dihydro-3,3-dimethyl-5-(methoxycarbonyl)-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-(methoxycarbonyl)-1-(4-sulfobutyl)-3H-indolium,sodium salt.

A-E: right lateral images, F: posterior image

A: prior to application,

B: 30 secs,

C: 1 min,

D: 10 mins,

E: 1 h after application,

F: 18 h after application.

1. An in-vivo diagnostic based on near infrared radiation, usingcompounds of the general formula IB_(L)−(F−W_(n))_(m)   (I) wherein L represents a number from 0 to 6, nrepresents a number from 0 to 10, and m represents a number from 1 to100; B is a biological detecting unit having a molecular weight of up to30,000 that bonds to specific cell populations or selectively toreceptors, or accumulates in tissues or tumours, or generally stays inthe blood, or is a macromolecule that bonds non-selectively; Frepresents a dye showing maximum absorption in the range of 650 to 1200nm; as well as their physiologically tolerable salts, characterized inthat F within the general formula I represents a cyanine dye of thegeneral formula IIa

wherein r represents the numbers 0, 1 or 2, wherein, for r=2, therespective L⁶ and L⁷ groups that occur in duplicate may be the same ordifferent, L¹ to L⁷ are the same or different, each independentlyrepresenting a CH or CR group, where R is a halogen atom, a hydroxy,carboxy, acetoxy, amino, nitro, cyano or sulfonic acid group or analkyl, alkenyl, hydroxyalkyl, carboxyalkyl, alkoxy, alkoxycarbonyl,sulfoalkyl, alkylamino, dialkylamino or halogenalkyl group containing upto 6 carbon atoms, an aryl, alkylaryl, hydroxyaryl, carboxyaryl,sulfoaryl, arylamino, diarylamino, nitroaryl or halogenaryl groupcontaining up to 9 carbon atoms, or where R represents a bond that bondsto another group R and forms a 4- to 6-member ring together with theinterspersed groups L¹ to L⁷, or two of L¹ to L⁷ that are linked via a—CO— group, R³ to R¹² are the same or different, each independentlyrepresenting a hydrogen atom, a group B as defined above or W as definedbelow, or an alkyl or alkenyl group containing up to 6 carbon atoms oran aryl or aralkyl group optionally carrying an additional group W asdefined below, or with 5- to 6- member rings that may be saturated,unsaturated or aromatic, and that may optionally carry an additionalgroup R as defined above, annelled to each pair of adjacent residue R³to R¹⁰ with due regard for the interspersed C atoms, X and Y are thesame or different, each independently representing an O, S, Se or Te ora —C(CH₃)_(2—), —CH=CH— or —CR¹³R¹⁴— group, where R¹³ and R¹⁴independently represent a hydrogen atom, a group B as defined above or Was defined below, or an alkyl or alkenyl group containing up to 6 carbonatoms or an aryl or aralkyl group containing up to 9 carbon atoms, thealkyl, alkenyl, aryl or aralkyl group optionally carrying an additionalgroup W as defined below, characterized in that W within the generalformula I is a carboxy or sulfonic acid group or a carboxyalkyl group oran alkoxycarbonyl or alkoxyoxoalkyl group containing up to 12 carbonatoms, and characterized in that W takes the position of R⁴ and R⁸and/or R⁶ and R¹⁰ and/or R¹¹ and R¹² .
 2. A method of in-vivo diagnosisof disease comprising administering at least one of the compounds ofclaim 1 to tissues and detecting said compound by means of near infraredradiation.
 3. An agent for in-vivo diagnosis comprising at least one ofthe cyanine dyes according to claim 1 together with common adjuvants,substrates and diluants.